Biomolecular modification of carbon nanotubes for studies of cell adhesion and migration

We report a strategy for tailoring and patterning carbon nanotubes (CNTs) for biospecific cell studies. We synthesized a new electroactive hydroquinone terminated pyrene molecule to tailor CNTs. These modified CNTs can be oxidized and chemoselectively reacted with oxyamine tethered ligands to generate various ligand tethered CNTs. A cell adhesive Arg-Gly-Asp peptide (RGD) is immobilized to the CNTs and a new microfluidic patterning method is employed to generate multiplex patterned surfaces for biospecific cell adhesion and migration studies. This work demonstrates the integration of a new functionalization strategy to immobilize a variety of ligands to CNTs for a range of potential drug delivery, tissue imaging and cellular behavior studies and a microfluidic patterning strategy for generating complex high-throughput surfaces for biotechnological and cell based assay applications.     

Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing

Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability, and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and the signaling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 mum when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization and initiate cell migration and signaling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.     

Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid （RGD） density effects on cell adhesion

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix （ECM）, being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid （RGD） density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy （AFM）, scanning tunneling microscopy （STM） and X-ray photoelectron spectroscopy （XPS）, showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout： Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions （FAs）. Therefore, dendrimer nano- patterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.     

Detection of the effect of environmental toxicants on the synthetic peptide modified neural cell chip

A cell based chip was designed to differentiate and to detect the effects of environmental chemicals on the neurite outgrowth in PC12 cell. To fabricate platform of cell chip, gold surfaces were modified by RGD based synthetic oligopeptide. Nanoscale controlled self-assembled peptide layer was investigated by Atomic Force Microscopy (AFM). On the fabricated cell chip, PC12 cell was immobilized and the differentiation of neurite outgrowth in PC12 cells was done by neurite growth factor (NGF). Differentiation of PC12 cell was confirmed by immunofluorescence study. Further the differentiation and the length of neurite was confirmed by confocal microscopy study. Voltammetry behavior of the neurite induced PC12 and the electrochemical behavior of the environmental toxicants effect on the neurite outgrowth was measured by cyclic voltammetry (CV). Self-assembled layer mediated cell immobilization technique and voltammetric signal analysis system can be applied to construct the neural cell chip for the detection of large number of environmental toxins and various neurotoxicants.     

Genetically Programmable Self‐Regenerating Bacterial Hydrogels

A notable challenge for the design of engineered living materials (ELMs) is programming a cellular system to assimilate resources from its surroundings and convert them into macroscopic materials with specific functions. Here, an ELM that uses Escherichia coli as its cellular chassis and engineered curli nanofibers as its extracellular matrix component is demonstrated. Cell‐laden hydrogels are created by concentrating curli‐producing cultures. The rheological properties of the living hydrogels are modulated by genetically encoded factors and processing steps. The hydrogels have the ability to grow and self‐renew when placed under conditions that facilitate cell growth. Genetic programming enables the gels to be customized to interact with different tissues of the gastrointestinal tract selectively. This work lays a foundation for the application of ELMs with therapeutic functions and extended residence times in the gut.     

Interface‐Directed Self‐Assembly of Cell‐Laden Microgels

Cell‐laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer‐scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter‐scale, cell‐laden hydrogels through the assembly of shape‐controlled microgels or a liquid–air interface. Cell‐laden microgels of specific shapes are randomly placed on the surface of a high‐density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue‐like structures. The resulting assemblies are cell‐laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air–liquid interface can be used to self‐assemble spatially controllable, cocultured tissue‐like structures.     

Engineering of Hydrogel Materials with Perfusable Microchannels for Building Vascularized Tissues

Vascular systems are responsible for various physiological and pathological processes related to all organs in vivo, and the survival of engineered tissues for enough nutrient supply in vitro. Thus, biomimetic vascularization is highly needed for constructing both a biomimetic organ model and a reliable engineered tissue. However, many challenges remain in constructing vascularized tissues, requiring the combination of suitable biomaterials and engineering techniques. In this review, the advantages of hydrogels on building engineered vascularized tissues are discussed and recent engineering techniques for building perfusable microchannels in hydrogels are summarized, including micromolding, 3D printing, and microfluidic spinning. Furthermore, the applications of these perfusable hydrogels in manufacturing organ‐on‐a‐chip devices and transplantable engineered tissues are highlighted. Finally, current challenges in recapitulating the complexity of native vascular systems are discussed and future development of vascularized tissues is prospected.     

Microfluidic Templating of Spatially Inhomogeneous Protein Microgels

3D scaffolds in the form of hydrogels and microgels have allowed for more native cell‐culture systems to be developed relative to flat substrates. Native biological tissues are, however, usually spatially inhomogeneous and anisotropic, but regulating the spatial density of hydrogels at the microscale to mimic this inhomogeneity has been challenging to achieve. Moreover, the development of biocompatible synthesis approaches for protein‐based microgels remains challenging, and typical gelation conditions include UV light, extreme pH, extreme temperature, or organic solvents, factors which can compromise the viability of cells. This study addresses these challenges by demonstrating an approach to fabricate protein microgels with controllable radial density through microfluidic mixing and physical and enzymatic crosslinking of gelatin precursor molecules. Microgels with a higher density in their cores and microgels with a higher density in their shells are demonstrated. The microgels have robust stability at 37 °C and different dissolution rates through enzymolysis, which can be further used for gradient scaffolds for 3D cell culture, enabling controlled degradability, and the release of biomolecules. The design principles of the microgels could also be exploited to generate other soft materials for applications ranging from novel protein‐only micro reactors to soft robots.     

Molding of hydrogel microstructures to create multiphenotype cell microarrays

The fabrication of mammalian cell-containing poly(ethylene glycol) (PEG) hydrogel microstructures on glass and silicon substrates is described. Using photoreaction injection molding in poly(dimethylsiloxane) microfluidic channels, three-dimensional hydrogel microstructures encapsulating cells (fibroblasts, hepatocytes, macrophage) were fabricated with cells uniformly distributed to each hydrogel microstructure, and the number of cells in each hydrogel microstructure was controlled by changing the cell density of the precursor solution. PEG hydrogels were modified using an Arg-Gly-Asp (RGD) peptide sequence, with the incorporation of RGD into the hydrogel matrix promoting the spreading of encapsulated fibroblasts over a 24-h period in culture. Cells remained viable encapsulated in these hydrogel microstructures for a period in excess of 1 week in culture. Arrays of hydrogel microstructures encapsulating two or more phenotypes on a single substrate were successfully fabricated using multimicrofluidic channels, creating the potential for multiphenotype cell-based biosensors.     

Multifunctional Transmembrane Protein Ligands for Cell‐Specific Targeting of Plasma Membrane‐Derived Vesicles

Liposomes and nanoparticles that bind selectively to cell‐surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single‐domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP‐tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering.     

Reduced Graphene Oxide‐GelMA Hybrid Hydrogels as Scaffolds for Cardiac Tissue Engineering

Biomaterials currently used in cardiac tissue engineering have certain limitations, such as lack of electrical conductivity and appropriate mechanical properties, which are two parameters playing a key role in regulating cardiac cell behavior. Here, the myocardial tissue constructs are engineered based on reduced graphene oxide (rGO)‐incorporated gelatin methacryloyl (GelMA) hybrid hydrogels. The incorporation of rGO into the GelMA matrix significantly enhances the electrical conductivity and mechanical properties of the material. Moreover, cells cultured on composite rGO‐GelMA scaffolds exhibit better biological activities such as cell viability, proliferation, and maturation compared to ones cultured on GelMA hydrogels. Cardiomyocytes show stronger contractility and faster spontaneous beating rate on rGO‐GelMA hydrogel sheets compared to those on pristine GelMA hydrogels, as well as GO‐GelMA hydrogel sheets with similar mechanical property and particle concentration. Our strategy of integrating rGO within a biocompatible hydrogel is expected to be broadly applicable for future biomaterial designs to improve tissue engineering outcomes. The engineered cardiac tissue constructs using rGO incorporated hybrid hydrogels can potentially provide high‐fidelity tissue models for drug studies and the investigations of cardiac tissue development and/or disease processes in vitro.     

Controlled release of drugs from gradient hydrogels for high-throughput analysis of cell-drug interactions

In this paper, we report a method to fabricate microengineered hydrogels that contain a concentration gradient of a drug for high-throughput analysis of cell-drug interactions. A microfluidic gradient generator was used to create a concentration gradient of okadaic acid (OA) as a model drug within poly(ethylene glycol) diacrylate hydrogels. These hydrogels were then incubated with MC3T3-E1 cell seeded glass slides to investigate the cell viability through the spatially controlled release of OA. The drug was released from the hydrogel in a gradient manner and induced a gradient of the cell viability. The drug concentration gradient containing hydrogels developed in this study have the potential to be used for drug discovery and diagnostics applications due to their ability to simultaneously test the effects of different concentrations of various chemicals.     

Role of Surface RGD Patterns on Protein Nanocages in Tumor Targeting Revealed Using Precise Discrete Models

The effectiveness of active targeting in cancer nanomedicine is becoming increasingly more debatable. Here, the role of the ligand functionalization patterns (number and distribution) on nanoparticle surfaces in tumor targeting is investigated using a 9 nm sized miniferritin protein nanocage, Dps modified with Arg‐Gly‐Asp (RGD) ligands whose functionalization patterns are precisely controlled. In vitro and in vivo experiments show that RGD modification endows Dps with tumor targeting capacity no matter what the surface pattern is. The tumor targeting of 2‐ligand Dps, which is better than that of 1‐ligand Dps, rivals or surpasses that of the 12‐ or 24‐ligand Dps. The 12‐ligand Dps with clustered RGD distribution shows 2.3 times the in vivo targeting efficiency of that with even distribution. The surface ligand pattern effects are correlated at least to receptor clustering and opsonization. This study provides insights into the understanding of the controversial findings on active tumor targeting in the literature and highlights the necessity of precise functionalization to achieve optimal active targeting in developing cancer nanomedicine.     

Independent Tuning of Nano-Ligand Frequency and Sequences Regulates the Adhesion and Differentiation of Stem Cells

The native extracellular matrix (ECM) can exhibit heterogeneous nano-sequences periodically displaying ligands to regulate complex cell–material interactions in vivo. Herein, an ECM-emulating heterogeneous barcoding system, including ligand-bearing Au and ligand-free Fe nano-segments, is developed to independently present tunable frequency and sequences in nano-segments of cell-adhesive RGD ligand. Specifically, similar exposed surface areas of total Fe and Au nano-segments are designed. Fe segments are used for substrate coupling of nanobarcodes and as ligand-free nano-segments and Au segments for ligand coating while maintaining both nanoscale (local) and macroscale (total) ligand density constant in all groups. Low nano-ligand frequency in the same sequences and terminally sequenced nano-ligands at the same frequency independently facilitate focal adhesion and mechanosensing of stem cells, which are collectively effective both in vitro and in vivo, thereby inducing stem cell differentiation. The Fe/RGD-Au nanobarcode implants exhibit high stability and no local and systemic toxicity in various tissues and organs in vivo. This work sheds novel insight into designing biomaterials with heterogeneous nano-ligand sequences at terminal sides and/or low frequency to facilitate cellular adhesion. Tuning the electrodeposition conditions can allow synthesis of unlimited combinations of ligand nano-sequences and frequencies, magnetic elements, and bioactive ligands to remotely regulate numerous host cells in vivo.     

Synthesis and characterization of matrix metalloprotease sensitive-low molecular weight hyaluronic acid based hydrogels

Hyaluronic acid is a naturally derived glycosaminoglycan (GAG) involved in biological processes. A low molecular weight hyaluronic acid (50 kDa)-based hydrogel was synthesized using acrylated hyaluronic acid. Matrix metalloproteinase (MMP) sensitive hyaluronic acid-based hydrogels were prepared by conjugation with two different peptides: cell adhesion peptides containing integrin binding domains (Arg-Gly-Asp: RGD) and a cross-linker with MMP degradable peptides to mimic the remodeling characteristics of natural extracellular matrices (ECMs) by cell-derived MMPs. Mechanical properties of these hydrogels were evaluated with different molecular weights of acrylated hyaluronic acid (10 kDa and 50 kDa) cross-linked by MMP sensitive peptides by measuring elastic modulus, viscous modulus, swelling ratio and degradation rate. The MMP sensitive hydrogel based on the 50 kDa hyaluronic acid showed a 31.5-fold shorter gelation time, 4.7-fold higher storage modulus and 0.51-fold smaller swelling ratio than those of the hydrogel based on the 10 kDa. Degradation rate was dependent on MMP sensitivity of the peptide cross-linker. MMP sensitive hyaluronic acid based hydrogels were degraded faster than MMP insensitive-hyaluronic acid-based hydrogels. Human mesenchymal stem cells (MSCs) were cultured in MMP-sensitive or insensitive hyaluronic acid-based hydrogels (50 kDa hyaluronic acid) and/or immobilized cell adhesive RGD peptides. Cells cultured in the MMP-sensitive hydrogel with RGD peptides showed dramatic cell spreading compared with that of the control, which remained round. This MMP-sensitive low molecular weight hyaluronic acid-based hydrogel could be useful in tissue engineering by improving tissue defect regeneration and tissue remodeling.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

Capillarity Guided Patterning of Microliquids

Soft lithography and other techniques have been developed to investigate biological and chemical phenomena as an alternative to photolithography‐based patterning methods that have compatibility problems. Here, a simple approach for nonlithographic patterning of liquids and gels inside microchannels is described. Using a design that incorporates strategically placed microstructures inside the channel, microliquids or gels can be spontaneously trapped and patterned when the channel is drained. The ability to form microscale patterns inside microfluidic channels using simple fluid drain motion offers many advantages. This method is geometrically analyzed based on hydrodynamics and verified with simulation and experiments. Various materials (i.e., water, hydrogels, and other liquids) are successfully patterned with complex shapes that are isolated from each other. Multiple cell types are patterned within the gels. Capillarity guided patterning (CGP) is fast, simple, and robust. It is not limited by pattern shape, size, cell type, and material. In a simple three‐step process, a 3D cancer model that mimics cell–cell and cell–extracellular matrix interactions is engineered. The simplicity and robustness of the CGP will be attractive for developing novel in vitro models of organ‐on‐a‐chip and other biological experimental platforms amenable to long‐term observation of dynamic events using advanced imaging and analytical techniques.     

Microfluidic‐Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high‐efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label‐free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor‐intensive steps of labeling molecular signatures of cells. In general, microfluidic‐based cell sorting approaches can separate cells using “intrinsic” (e.g., fluid dynamic forces) versus “extrinsic” external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label‐free microfluidic‐based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic‐based cell separation methods are discussed.     

Solvent‐Assisted Micromolding of Biohybrid Hydrogels to Maintain Human Hematopoietic Stem and Progenitor Cells Ex Vivo

Array‐format cell‐culture carriers providing tunable matrix cues are instrumental in current cell biology and bioengineering. A new solvent‐assisted demolding approach for the fabrication of microcavity arrays with very small feature sizes down to single‐cell level (3 µm) of very soft biohybrid glycosaminoglycan–poly(ethylene glycol) hydrogels (down to a shear modulus of 1 kPa) is reported. It is further shown that independent additional options of localized conjugation of adhesion ligand peptides, presentation of growth factors through complexation to gel‐based glycosaminoglycans, and secondary gel deposition for 3D cell embedding enable a versatile customization of the hydrogel microcavity arrays for cell culture studies. As a proof of concept, cell‐instructive hydrogel compartment arrays are used to analyze the response of human hematopoietic stem and progenitor cells to defined biomolecular and spatial cues.